The pathogenic fungus Cryptococcus neoformans has as its main virulence factor an immense capsule that is largely composed of two polysaccharides that both contain xylose residues bound to alpha-1, 3 linked mannose. Previous studies have shown that a lack of these xylose residues yields the organism avirulent and that changes in the linkages between mannose and xylose can alter the level of virulence. However, little is known about how this organism incorporates xylose into these glycans. As described in Aim I, we have developed an assay for xylosyltransferase activity that will be used to purify a protein with activity appropriate for the transfer of xylose to the capsule polysaccharides. I will obtain protein sequence and characterize this enzyme, including determination of the linkage formed within the product. In Aim II, I will clone the corresponding gene, express the protein and then interrupt the xylosyltransferase activity with RNA interference and/or gene disruption. The capsular structure of the strains with altered xylosyltransferase activity will be characterized, along with the ability of these strains to cause disease in a mouse model of virulence. The information obtained will guide future studies to fully elucidate the process of xylose transfer.